Study On The Function Of PA0335 In Pseudomonas Aeruginosa PAO1

Pseudomonas aeruginosa(PA)is one of the most widely recognized opportunistic Gram-negative pathogenic bacteria,causing considerable morbidity and mortality in humans and animals.In recent years,the survivability and tolerance of PA are continuously increasing in low-nutrient and other stress external conditions.Amino acids play a crucial role on the stress survive and intracellular regulatory in PA.A transposition mutant library has been previously screened for the biodegradation-related genes in Pseudomonas aeruginosa PAO1.A transposon mutant E9 showed inhibited growth on a minimal medium.The insert site locates in125 bp of PA0334,opposited to the operon.However,the inhibited growth of Tn0334 was not due to the function loss of PA0334.This study focus on the identification and characterization of the key gene responble for the phenotype of Tn0334.First,the deletion mutant PAO1(?PA0334-5)and the complement were constructed.The key gene PA0335 was determined to be responble for the inhibited growth of Tn0334 on the minimal medium.Therefore,the deletion mutant and complement of PA0335 were constructed.This study uses the method of auxanograph and growth investigation on the different kinds of medium,PA0335 was found to be related to the incomplete histidine auxotrophs because the mutant PAO1(?PA0335)could barely growth in the minimal medium without histidine in first 3 day and growed poorly 3day later.PA0335 thus has been found to be involved in intracellular L-histidine biosynthesis.The growth recovery of PAO1(?PA0335)after 3 days indicated that there could probably exist other genes with the similar function to PA0335 in PAO1.The growth of PAO1(?PA0335)in he strain will become small-colony variants(SCVs)through rigorous control of the reaction.PAO1(?PA0335)when PAO1(?PA0335)responds to environmental stress.PA0335 belong to the HAD superfamily according to the bioinformatics analysis.PA0335 has been confirmd to encode one kind of alkaline phosphatases(EC 3.1.3.-)which could catalyze the dephosphorylation of the substrate because the in vitroexpressed protein showed obvious phosphatase activity.Furthermore,a random transposition mutant library containing 16249 clones was constructed in the background of PAO1(?PA0335)and screened for the mutants showing recoved growth in minimal medium.The mutatanted genes were confirmed by RT-PCR and the sequence analysis for the five right transposition mutants.The inserted genes are all related to PST system.Therefore PA0335 was kind of alkaline phosphatases.The deletion of PA0335 resulted in incomplete histidine auxotrophs of PAO1.